Sequence studies on pyridoxal phosphate-dependent enzymes

The amino acid sequences of two pyridoxal phosphate-dependent enzymes, namely glutamate decarboxylase and 4-aminobutyrate aminotransferase, were solved by a combination of direct protein sequencing and cDNA sequencing. In the case of glutamate decarboxylase from Escherichia coli, correct ordering of three internal peptides was achieved by sequencing a fragment of DNA generated by PCR. In the case of 4-aminobutyrate aminotransferase from pig liver, chemical studies on the reaction of the enzyme with a suicide inhibitor allowed a detailed description of the mechanism of inactivation and collection of partial sequence information on the protein. The sequence of the protein was completely reconstructed after analysis of further peptide fragments derived by proteolytic and chemical cleavages, with the help of the information from the cDNA-deduced sequence of pig brain 4-aminobutyrate aminotransferase performed by another group (Kwon, Park and Churchich [1992] J. Biol. Chem. 267, 7215-7216).